Methods And Compositions Relating to Viral Latency

ABSTRACT

Disclosed are compositions and methods that relate generally to viruses, and more particularly to the agents and their identification and use of anti-HIV agents which cause latently infected cells to reactivate.

This application claims the benefit of U.S. Provisional Application No.61/147,649, filed on Jan. 27, 2009, which is incorporated by referenceherein in its entirety.

This invention was made with government support under Grant AI49057awarded by the NIH NIAID. The government has certain rights in theinvention.

BACKGROUND

The advent of highly active antiretro viral therapy (HAART), whichinvolves the use of three or more antiretroviral drugs, has led to asignificant improvement in the care and survival of patients infectedwith HIV-I . In patients not infected with resistant strains of thevirus, HAART typically results in a dramatic decrease in viral loadoften from levels of 10,000-100,000 RNA copies/ml of plasma to less than50 copies/ml.

Given the dramatic effects of HAART, it was proposed that completeelimination of the virus might be possible within 2 to 3 years. However,even after long-term suppression of viral replication with HAART, thevirus rapidly rebounds after therapy is discontinued. A key contributorto viral rebound appears to be a reservoir of latently+infected cells,including CD4 memory T cells. The half-life of the latently infectedpopulation is quite long, and it is estimated that it would take over 60years of HAART to eliminate this population. Therefore, life-long HAARTwould be required to control infection in patients.

Retroviruses, including HIV-I, are RNA viruses that replicate through aDNA intermediate and integrate very efficiently into the genome of aninfected cell forming a pro virus. Once the pro virus is formed, it ismaintained in the genome of the infected cell and transferred todaughter cells in the same fashion as any other genetic element withinthe cellular genome. Thus, the virus has the potential to persist if itinfects long-lived cells such as memory T cells. It has been known since1986 that HIV-I can establish a latent infection in culture. It wasfound that a human T cell line infected with replication-competent viruscould develop a latent infection in which the provirus was dormant butcould be reactivated upon stimulation. Since then it has beenestablished that a number of cytokines can reactivate latent proviruses.

The role that latency is playing in preventing clearance of the virusinfection has become evident in recent years. Patients that had beensuccessfully treated with HAART in which viral RNA was maintained atlevels below 50 copies/ml in the plasma for years, experienced rapidvirus rebound upon withdrawal of therapy. Moreover, it was found thatafter T cell activation, virus could be isolated from CD4 T cells takenfrom these patients making it clear that to eradicate the virus it willbe necessary to eliminate the latently infected cells.

There have been attempts to flush the latent virus from infectedindividuals by nonspecific activation of T cells to “turn on” latentproviruses. As part of this approach, the patients remain on HAART toprevent new infections, and the infected cells from which the latentproviruses are activated should die due to cytotoxic effects of viralexpression and/or because of targeting by the immune system which canrecognize the cells once they begin to express the viral proteins.

Thus, there is a need in the art for further strategies to discover newdrugs capable of activating latent viruses.

SUMMARY

Disclosed are methods for creating a population of cells latentlyinfected with a virus comprising the steps of a) isolating primarycells; b) priming the cells toward differentiation, wherein at least aportion of primary cells differentiate into non-polarized cells; c)exposing the non-polarized cells of step b) to a virus defective in Env;thereby creating a population of cells latently infected with a virusand wherein the Env is provided in trans to the env defective viruswhile the virus is being grown, prior to exposure to the non-polarizedcells of step c.

Also disclosed herein are cell lines comprising non-polarized CD4+ cellsthat have been latently infected with a virus.

Also disclosed are methods of reactivating a cell latently infected withvirus, the method comprising activating NFAT in the absence of NFκB.

Also disclosed are methods of reactivating a cell latently infected withvirus, the method comprising contacting the cell with IL-7 in theabsence of NFAT.

Disclosed herein are methods of treating a subject with a retrovirus,the method comprising: a) exposing the subject to a composition thatreactivates cells latently infected with a retrovirus; and b) treatingthe subject with an antiretroviral agent identified by a methoddisclosed herein.

Also disclosed are methods of screening for a composition that activatesa cell latently infected by a virus; the method comprising the steps of:a) creating a latently infected cell; b) exposing the cell to a testcomposition; and c) determining if the latently infected cell becomesactive.

Further disclosed are compositions identified by the screening methoddescribed above.

Also disclosed is an assay for determining a composition capable ofreactivating a cell latently infected with a retrovirus, the assaycomprising an in vitro population of cells latently infected with aretrovirus, wherein at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60%, or more of the cells are latently infected, and wherein the cellpopulation is stable.

Also disclosed are kits for screening for compositions that reactivate alatently infected virus in a cell comprising cytokines, latentlyinfected cells, and antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description illustrate the disclosed compositions and methods.

FIG. 1 shows a model of HIV-1 latency. Procedure used for the generationof human primary memory T cells and subsequent establishment of latentinfections.

FIG. 2 shows a generation of Latently HIV-1 infected primary CD4⁺ Tcells ex vivo. Cells were primed in NP, Th1- or Th2-polarizingconditions and 7 days after activation cells were infected with DHIV.(A) 3 and 5 days p.i. cells were assessed for intracellular p24 gagexpression by flow cytometry. The percentage of p24-positive cells isindicated in each panel. The experiment shown is representative of 4different experiments with 4 different donors. (B) 5 days p.i. cellswere assessed for annexin-PE and DiOC₆(3) by flow cytometry. For eachpanel, the percentage of apoptotic cells (annexin-PE positive andDiOC₆(3) low) is indicated. The experiment shown is representative of 3different experiments with 3 different donors (C) 7 days after infectioncells were cultured without stimulation (untreated) or co-stimulatedwith antibodies to CD3 and CD28 for 3 days (CD3/CD28) and assessed forintracellular p24 gag expression by flow cytometry. The percentage ofp24-positive cells is indicated in each panel for this representativeexperiment. Values corresponding to 7 different donors are shown in (D),where each symbol represents a different donor and horizontal linesindicate media values. Significance by two-tailed Paired-Samples T testanalysis (p values provided). (E) Viral integration was analyzed byAlu-PCR 3 days p.i. in Donors 1 and 2. Horizontal lines indicate medianvalues.

FIG. 3 shows the signaling pathways leading to HIV-1 reactivation I. NPcells were infected with DHIV and 7 days after infection cells were leftuntreated or co-stimulated with antibodies to CD3 and CD28 for 3 days(CD3/CD28) in the presence of the indicated inhibitor for the protein orpathway indicated between parentheses and assessed for intracellular p24gag expression by flow cytometry. (A) Representative experiment. Thepercentage of p24-positive cells is indicated in each panel. (B)Box-plots corresponding to 3 different donors. Horizontal lines indicatemedian values and significance by two-tailed Paired-Samples T testanalysis (p values provided).

FIG. 4 shows signaling pathways leading to HIV-1 reactivation II. NPcells were infected with DHIV and 7 days after infection cells were leftuntreated, co-stimulated (CD3/CD28) or in the presence of the indicatedagonist for the protein or pathway indicated between parentheses for 3days and assessed for intracellular p24 gag expression by flowcytometry. In the case of cells stimulated with PHA, cells were alsoco-stimulated in the presence of the inhibitors PP2 (Lck) or CsA (NFAT).(A) Representative experiment. The percentage of p24-positive cells isindicated in each panel. (B) Box-plots corresponding to 3 differentdonors. Horizontal lines indicate median values and significance bytwo-tailed Paired-Samples T test analysis (p values provided).

FIG. 5 shows the transcription factor binding sites involved in HIV-1reactivation. (A) Scheme of HIV-1 LTR. (B) NP cells were infected withwt DHIV or with different LTR mutants. Mutations can be viewed in FIG.7. 7 days after infection cells were co-stimulated with antibodies toCD3 and CD28 for 3 days and assessed for intracellular p24 gagexpression by flow cytometry. The percentage of p24-positive cells isindicated in each panel. Percentage of viral integration by Alu-PCR foreach virus is indicated in blue (UD=undetectable). The experiment isrepresentative of 3 different experiments with 3 different donors.

FIG. 6 shows Phenotypic analysis of T cells. Naïve cells were primed inNP, Th1-or Th2-polarizing conditions and were subject to an extensivelyphenotypic analysis. Data are representative of analysis performed with5 different donors. (A) Activation of naïve T cells led to expression ofthe activation markers, CD69, CD25 and HLA-DR: CD69 (early activationmarker) and CD25 (medium-time activation marker) were analyzed 3 daysafter activation. HLA-DR (late activation marker) was analyzed 7 daysafter activation. (B) HIV-1 receptor, CD4, and co-receptors, CXCR4 andCCR5, were analyzed at the time of infection (7 days after activation)and compared with naïve cells. (C) CD45RA and CD45RO were analyzed atthe time of infection and compared with naïve cells. The subset analyzedby each marker is indicated between parentheses. (D) Cells were analyzedfor the expression of CCR7 and CD27, surface markers expressed in naïveand central memory T cells at day 0, 7, 14 and 21-post activation. (E)The phenotype of Th1 and Th2 cells was confirmed via intracellularstaining for IFN-γ and IL-4, respectively 7 days after activation. Onday 7, cells were restimulated with PMA plus Ionomycin for 1 h plus anadditional 3 h in the presence of brefeldin A for intracellular cytokinedetection. Also, cells were analyzed for the expression of CrTH2,surface marker expressed in Th2.

FIG. 7 shows p24 Gag intracellular staining correlates with GFPexpression. Cells were primed in NP conditions and 7 days afteractivation cells were non-infected (Mock), infected with DHIV (DHIVInfected) or infected with a DHIV in which nef has been replaced by GFP(DHIV-GFP Infected). 3 days after infection cells were assessed forintracellular p24 Gag and GFP expression by flow cytometry. 7 days afterinfection cells were cultured without stimulation (untreated) orco-stimulated with antibodies to CD3 and CD28 for 3 days (CD3/CD28) andassessed for intracellular p24 Gag and GFP expression by flow cytometry.The percentage of cells is indicated in each panel for thisrepresentative experiment.

FIG. 8 shows T cell signaling. Diagram to describe the main T signalingpathways analyzed in this work. The inhibitors and agonist used in thiswork are represented with red letters or green letters, respectively.

FIG. 9 shows a panel of LTR mutants. Scheme representing the differentmutants generated within the HIV-1 LTR. In each one, the nucleotidesmutated are represented with bold letters.

FIG. 10 shows that stimulation by IL-2+IL-7 induces viral reactivationin both dividing and non-dividing cells.

FIG. 11 shows that stimulation by IL-7 can lead to viral reactivation bytwo separate signaling pathways.

FIG. 12 shows the percentage of reactivation by either αCD3/αCD28 orIL2/1L7 stimulation following administration of an inhibitor of p38(SB202190) or NFAT (CsA).

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10″as well as “greater than orequal to 10” is also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

By “inducible expression system” is meant a construct or combination ofconstructs that includes a nucleotide sequence encoding atransactivator, an inducible promoter that can be transcriptionallyactivated by the transactivator, and a nucleotide sequence of interestoperably linked to the inducible promoter.

By “transactivator,” “transactivating factor,” or “transcriptionalactivator” is meant a polypeptide that facilitates transcription from apromoter. Where the promoter is an inducible promoter, thetransactivator activates transcription in response to a specifictranscriptional signal or set of transcriptional signals.

By “envelope protein” is meant a polypeptide that 1) can be incorporatedinto an envelope of a virus such as a retrovirus; and 2) can bind targetcells and facilitate infection of the target cell by the RNA or DNAvirus that it envelops. “Envelope protein” is meant to includenaturally-occurring (i.e., native) envelope proteins and functionalderivatives thereof that 1) can form pseudotyped retroviral virions, and2) exhibit a desired functional characteristic(s) (e.g, facilitate viralinfection of a desired target cell, and/or exhibit a different oradditional biological activity) when provided in trans. Such envelopeproteins include retroviral envelope proteins derived from any suitableretrovirus (e.g., an amphotropic, xenotropic, ecotropic or polytropicretrovirus) as well as non-retroviral envelope proteins that can formpseudotyped retroviral virions (e.g., VSV G). Envelope proteins ofparticular interest include, but are not limited to, envelope protein ofvesicular stomatis virus (VSV G), HTLV-1, gibbon ape leukemia virus(GALV), Sindai virus, influenza virus, herpes virus, rhabdovirus, andrabies virus.

By “functional derivative of a polypeptide” is meant an amino acidsequence derived from a naturally-occurring polypeptide that is alteredrelative to the naturally-occurring polypeptide by virtue of addition,deletion, substitution, or other modification of the amino acidsequence. “Functional derivatives” contemplated herein exhibit thecharacteristics of the naturally-occurring polypeptide essential to theoperation of the invention.

By “promoter” is meant a minimal DNA sequence sufficient to directtranscription of a DNA sequence to which it is operably linked. The term“promoter” is also meant to encompass those promoter elements sufficientfor promoter-dependent gene expression controllable for cell-typespecific expression, tissue-specific expression, or inducible byexternal signals or agents; such elements may be located in the 5′ or 3′regions of the naturally-occurring gene.

By “inducible promoter” is meant a promoter that is transcriptionallyactive when bound to a transcriptional activator, which in turn isactivated under a specific condition(s), e.g., in the presence of aparticular chemical signal or combination of chemical signals thataffect binding of the transcriptional activator to the induciblepromoter and/or affect function of the transcriptional activator itself.

By “construct” is meant a recombinant nucleotide sequence, generally arecombinant DNA molecule, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences. In general,“construct” is used herein to refer to a recombinant DNA molecule.

By “operably linked” is meant that a DNA sequence and a regulatorysequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules (e.g., transcriptional activatorproteins) are bound to the regulatory sequence(s).

By “operatively inserted” is meant that a nucleotide sequence ofinterest is positioned adjacent a nucleotide sequence that directstranscription and translation of the introduced nucleotide sequence ofinterest (i.e., facilitates the production of, e.g., a polypeptideencoded by a DNA of interest).

By “packaging cell line” is meant a line of packaging cells selected fortheir ability to package defective retroviral vectors at a titer ofgenerally greater than 10³ virions per milliliter of tissue culturemedium, having less than 10 helper virus virions per milliliter oftissue culture medium, and capable of being passaged in tissue culturewithout losing their ability to package defective retroviral vectors.

By “transformation” is meant a permanent or transient genetic change,preferably a permanent genetic change, induced in a cell followingincorporation of new DNA (i.e., DNA exogenous to the cell). Where thecell is a mammalian cell, a permanent genetic change is generallyachieved by introduction of the DNA into the genome of the cell.

By “target cell” is meant a cell(s) that is to be transformed using themethods and compositions of the invention. Transformation may bedesigned to non-selectively or selectively transform the target cell(s).

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding a gene product (e.g., RNA and/or protein) ofinterest (e.g., nucleic acid encoding a therapeutic cellular product).

By “subject” or “patient” is meant any subject for which celltransformation or gene therapy is desired, including humans, non-humanprimates, cattle, dogs, cats, guinea pigs, rabbits, mice, insects,horses, chickens, and any other genus or species having cells that canbe infected with a viral vector having an envelope containing VSV G orother envelope described herein.

By “transgenic organism” is meant a non-human organism (e.g.,single-cell organisms (e.g., yeast), mammal, non-mammal (e.g., nematodeor Drosophila)) having a non-endogenous (i.e., heterologous) nucleicacid sequence present as an extrachromosomal element in a portion of itscells or stably integrated into its germ line DNA.

By “transgenic animal” is meant a non-human animal, usually a mammal,having a non-endogenous (i.e., heterologous) nucleic acid sequencepresent as an extrachromosomal element in a portion of its cells orstably integrated into its germ line DNA (i.e., in the genomic sequenceof most or all of its cells). Heterologous nucleic acid is introducedinto the germ line of such transgenic animals by genetic manipulationof, for example, embryos or embryonic stem cells of the host animal.

By “viral vector” is meant a recombinant viral particle thataccomplishes transformation of a target cell with a nucleotide sequenceof interest.

By “virion,” “viral particle,” or “retroviral particle” is meant asingle virus minimally composed of an RNA or DNA genome, Pol protein(for reverse transcription of the RNA genome following infection), Gagprotein (structural protein present in the nucleocapsid), and anenvelope protein. As used herein, the RNA genome of the retroviralparticle is usually a recombinant RNA genome, e.g., contains an RNAsequence exogenous to the native retroviral genome and/or is defectivein an endogenous retroviral sequence (e.g., is defective in pol, gag,and/or env, and, as used herein, is normally defective in all threegenes).

By “pseudotyped viral particle,” or “pseudotyped retroviral particle” ismeant a viral particle having an envelope protein that is from a virusother than the virus from which the RNA genome is derived. The envelopeprotein can be from a retrovirus of a species different from theretrovirus from which the RNA genome is derived or from a non-retroviralvirus (e.g., vesicular stomatitis virus (VSV)).

By “VSV G” or “VSV G envelope protein” is meant the envelope protein ofvesicular stomatitis virus (VSV) or a polypeptide derived therefrom orrecombinant fusion polypeptide having a VSV G polypeptide sequence fusedto a heterologous polypeptide sequence, where the VSV G-derivedpolypeptide of recombinant fusion polypeptide can be contained in aviral envelope of a pseudotyped retroviral particle and retainsinfectivity for a desired target cell (e.g., a range of desiredeukaryotic cells, or a specific target cell of interest).

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

Although embodiments have been depicted and described in detail herein,various modifications, additions, substitutions and the like can bemade.

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular notch structural motif is disclosed anddiscussed and a number of modifications that can be made to a number ofmolecules including the notch structural motif are discussed,specifically contemplated is each and every combination and permutationof notch structural motif and the modifications that are possible unlessspecifically indicated to the contrary. Thus, if a class of molecules A,B, and C are disclosed as well as a class of molecules D, E, and F andan example of a combination molecule, A-D is disclosed, then even ifeach is not individually recited each is individually and collectivelycontemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E,and C-F are considered disclosed. Likewise, any subset or combination ofthese is also disclosed. Thus, for example, the sub-group of A-E, B-F,and C-E would be considered disclosed. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed compositions. Thus, if thereare a variety of additional steps that can be performed it is understoodthat each of these additional steps can be performed with any specificembodiment or combination of embodiments of the disclosed methods.

B. Compositions and Methods

The use of antiretroviral therapy in human immunodeficiency virus type 1(HIV-1) infected patients does not lead to virus eradication. This isdue, to a significant degree, to the fact that HIV-1 can establish ahighly stable reservoir of latently infected cells. In this work, an exvivo experimental system that generates high levels of HIV-1 latentlyinfected memory cells using primary CD4+ T cells is described. Use ofthis model enabled the dissection of the T cell-signaling pathways andcharacterization of the long terminal repeat (LTR) cis-acting elementsinvolved in reactivation of HIV-1 in memory CD4+ T cells. The results ofthis study conclude that Lck and NFAT are required for optimal latentvirus reactivation in memory T cells. It was also found that thecis-acting elements which are critical toward HIV-1 reactivation are theSp1 and κB/NFAT transcription factor binding sites.

HIV-1 persists in infected individuals even in the presence of HAART.The principal reservoir of HIV-1 latency is thought to reside inresting, CD4⁺ memory T cells, which harbor integrated HIV-1(Finzi et al.1997). The low frequency of latently infected cells (1 in 10⁶ restingCD4⁺ T cells (Chun et al. 1997)), for which known phenotypic markers arenot available, poses a great challenge to the study of latency in vivo.

Previous studies on HIV-1 latency were based on the generation ofchronically infected cell lines, such as the ACH2 (Folks et al. 1989),JΔK (Antoni et al. 1994), and J-Lat (Jordan et al. 2003) T-cell lines,and the U1 promonocytic cell line (Folks et al. 1987). In these systems,latency was defined as a state in which integrated proviruses failed todrive efficient gene expression. However, these systems do notnecessarily reflect the latency state in vivo because the lack of viralgene expression is due to mutations in that (ACH2 and U1 (Folks et al.1989, Folks et al. 1987)) or mutations in the LTR (JΔK T-cell line(Antoni et al. 1994)). While these latency models recapitulate aplethora of mechanisms that can underlie viral latency, the focus ofthis study was in developing a more general model that did not rely onclonal proviral integration sites, and which utilized non-transformed,primary human T-cells.

Recently, a model using human fetal liver tissue in SCID-hu mice hasgenerated a great deal of interest in the field of HIV-1 latency (Brookset al. 2001). This model relies upon infection of thymocytes and thevast majority of latently infected cells in this system are mature,quiescent CD4⁺ single positive naïve T cells. This is in contrast withfindings in HIV-1 patients, where the majority of latently infectedcells are CD4⁺ memory T cells (Finzi et al. 1997). Although naïve andmemory cells, share the characteristic of being quiescent, a likelyrequirement for HIV-1 latency in T cells (Finzi et al. 1997), there areimportant differences between these cell types that impact latency andreactivation.

Disclosed herein is the development of a novel HIV-1 latency andreactivation models that use human, primary cells. This model is used todissect relevant signaling pathways involved in viral reactivation fromlatently infected memory CD4⁺cells.

1.Cell Cultures and Methods Thereof

In order to screen for agents that can reactivate latent virus in acell, it is necessary to have a latently infected cell. While it ispossible to conduct such screening in vivo, the ability to controlreactivation is limited in said situations and can be very expensive. Anin vitro method of screening for agents that induce reactivation oflatently infected virus avoids the problems of in vivo systems and isvastly lest expensive. However, to create such a system requires thepresence of a latently infect cell line. Therefore, disclosed herein isa method for creating a population of cells latently infected with avirus, the method comprising the steps of: a) isolating primary cells;b) priming the cells toward differentiation, wherein at least a portionof primary cells differentiate into non-polarized cells; c) exposing thenon-polarized cells of step b) to a virus defective in Env; therebycreating a population of cells latently infected with a virus andwherein the Env is provided in trans to the env defective virus whilethe virus is being grown, prior to exposure to the non-polarized cellsof step c.

Examples of retroviral-derived env genes which can be employed hereininclude, but are not limited to type C retroviral envelope proteins,such as those from Moloney murine leukemia virus (MoMuLV), Xenotropicmurine leukemia virus-related virus (XMRV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus(GaLV), and Rous Sarcoma Virus (RSV). Other viral env genes which can beused include, for example, env genes from immunodeficiency viruses(HIV-1, HIV-2, FIV, SIV and EIV), human T cell leukemia viruses (HTLV-1,HTLV-2, HTLV-3 and HTLV-4), herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV,HHV-6, HHV-7, HHV-8), and Vesicular stomatitis virus (VSV) (Protein G).When producing recombinant retroviruses of the invention (e.g.,recombinant lentiviruses), the wild-type retroviral (e.g., lentiviral)env gene can be used, or can be substituted with any other viral envgene, such those listed above. Methods of pseudotyping recombinantviruses with envelope proteins from other viruses in this manner arewell known in the art. As referred to herein, a “pseudotype envelope” isan envelope protein other than the one that naturally occurs with theretroviral core virion, which encapsidates the retroviral core virion(resulting in a phenotypically mixed virus).

Viral envelope proteins of the invention (whether pseudotyped or not)can also be modified, for example, by amino acid insertions, deletionsor mutations to produce targeted envelope sequences such as ecotropicenvelope with the EPO ligand, synthetic and/or other hybrid envelopes;derivatives of the VSV-G glycoprotein. Furthermore, it has been shownthat it is possible to limit the infection spectrum of retroviruses andconsequently of retroviral-based vectors, by modifying the viralpackaging proteins on the surface of the viral particle (see, forexample PCT publications WO93/25234 and WO94/06920). For instance,strategies for the modification of the infection spectrum of retroviralvectors include: coupling antibodies specific for cell surface antigensto the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Julan etal. (1992) J. Gen Virol 73:3251-3255; and Goud et al. (1983) Virology163:251-254); or coupling cell surface receptor ligands to the viral envproteins (Neda et al. (1991) J Biol Chem 266:14143-14146). Coupling canbe in the form of the chemical cross-linking with a protein or othervariety (e.g. lactose to convert the env protein to anasialoglycoprotein), as well as by generating fusion proteins (e.g.single-chain antibody/env fusion proteins).

The primary cells can be lymphocytes (e.g., CD4, CD8, and B cells),macrophages, dendritic cells, neurons, or epidermal cells. For example,the primary cell can be a CD4+ T-cell, such as naïve CD4 T-cells or amemory CD4+ T-cells (e.g., T_(CM)). It is understood that the type ofprimary cell depends on the latent reservoir of the virus for whichreactivation is sought. Thus, for example, for HIV-1 or HIV-2, theprimary cell is a CD4 T cell. By contrast, for EBV, the primary cell isa B cell and for HSV-1 and HSV-2, the primary cell is a neuron. It isunderstood that those of skill in the art will know the appropriateprimary cell to establish latency for the given virus.

The virus can be any virus capable of producing latently infected cells.This includes, but is not limited to, retroviruses such as HIV-1, HIV-2,SIV, XMRV, HTLV-1, HTLV-2, HTLV-3 and HTLV-4 and herpesviruses such asHerpes Simplex virus 1 (HSV-1 also known as HHV-1), Herpes Simplexvirus-2 (HSV-2 also known as HHV-2), Varicella Zoster virus (VZV);Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6(HHV-6), Human Herpes virus-7 (HHV-7 also referred to as Roseolovirus),and Human Herpes virus-8 (HHV-8 also referred to as Karposi's sarcomaassociated herpesvirus). The virus can also be hepatitis B or hepatitisC.

Retroviruses are enveloped RNA viruses that, after infection of a hostcell, reverse transcribe their RNA genomes into a DNA intermediate, orprovirus. All viruses containing an RNA genome and producing anRNA-dependent DNA polymerase are contained in the retroviral family. Thefamily is divided into three subfamilies: (1) Oncovirinae, including allthe oncogenic retroviruses, and several closely related non-oncogenicviruses; (2) Lentivirinae, the “slow retroviruses” such as the humanimmunodeficiency virus (HIV) and visna virus; and (3) Spumavirinae, the“foamy” retroviruses that induce persistent infections, generallywithout causing any clinical disease. Retroviruses containing at leastthree types of proteins encoded by the viral genome, i.e., gag proteins(the group antigen internal structural proteins), pol proteins (theRNA-dependent DNA polymerase and the protease and integrase proteins),and env proteins (the viral envelope protein or proteins). In additionto genes encoding the gag, pol, and env proteins, the genome to theretrovirus includes two long terminal repeat (LTR) sequences, one at the5′ and one at the 3′ end of the virus. These 5′ and 3′ LTRs promotetranscription and polyadenylation of viral mRNAs and participate in theintegration of the viral genome into the cellular DNA of the host.

In the methods disclosed herein, a significant percentage of thepopulation of infected cells can be latently infected. At least 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60% or more of the population ofcells can be latently infected.

Also disclosed herein is a cell line comprising non-polarized CD4+ cells(e.g., naïve or T_(CM) cells) that have been latently infected with avirus. Similarly disclosed herein are cell lines comprising B cells, CD8T cells (naïve or memory), macrophages, hepatocytes, epidermal cells, orneurons latently infected with a virus.

The virus can be any virus capable of producing latently infected cells.This includes, but is not limited to, retroviruses such as HIV-1, HIV-2,SIV, XMRV, HTLV-1, HTLV-2, HTLV-3 and HTLV-4. The virus can also beHSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8, hepatitis C, orhepatitis B.

Also disclosed is a method of reactivating a cell latently infected withvirus, the method comprising activating NFAT in the absence of NF-κB.Alternatively, also disclosed are methods of reactivating a celllatently infected with virus, the method comprising contacting a cellwith IL-7 in the absence of NFAT. The reactivation can utilize thep38/MAP Kinase pathway. However, disclosed herein, the utilization ofthe p38/MAPK pathway can vary depending on the initial stimulus forreactivation. For example, in the case of a T cell, where reactivationis induced through stimulation of the TCR, then the p38/MAPK pathwayinvolving NFAT is utilized. By contrast, reactivation of a latentlyinfected cell via the use of IL-7 would use a p38/MAPK pathway that doesnot involve NFAT.

The virus can be any virus capable of producing latently infected cells.This includes, but is not limited to, retroviruses such as HIV-1, HIV-2,SIV and HTLV. The virus can also be hepatitis B or hepatitis C. NFAT canalso be activated in the presence of CD3/CD28, or for example, can beactivated in the presence of CD3/CD28 and Sp1.

The virus can be any virus capable of producing latently infected cells.This includes, but is not limited to, retroviruses such as HIV-1, HIV-2,SIV, XMRV, HTLV-1, HTLV-2, HTLV-3 and HTLV-4. The virus can also beHSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-8, hepatitis C, or hepatitis B.

Disclosed herein is a method of treating a subject with a retrovirus,the method comprising: a) exposing the subject to a composition thatreactivates cells latently infected with a retrovirus; and b) treatingthe subject with an antiretroviral agent identified by a methoddisclosed herein.

As described above, highly active antiretroviral therapy (HAART) has hadan important impact upon morbidity and mortality from AIDS. AlthoughHAART results in a remarkable suppression of HIV-I replication ininfected patients, it does not provide for elimination of the virus evenafter years of suppressive therapy. Complete viral clearance cannot beachieved due to the presence of latently infected cells in patients,which upon withdrawal of HAART, contribute to viral rebound. Attempts ateradicating latently infected cells by activating them with cytokinesand lymphokines has not met with success probably owing both to theinability of this treatment to reach all of the latent viral reservoirsand to the toxicity of the regimen. Small molecules with pharmacologicalproperties that allow them to reach all viral reservoirs and activatelatent HTV-I pro viruses result in clearance of HIV-I infections whenused in combination with HAART.

Disclosed herein is a latently infected cell line that can be used forhigh throughput screening (HTS) to identify small molecules that can beemployed to eradicate latent virus from infected individuals.

2. Methods of Screening

The potential for viral latency to maintain a viral reservoir in asubject undergoing antiviral treatment necessitates that the subjectnever stops taking antivirals. By reactivating the virus whilemaintaining antiviral treatment, the viral reservoir can be depleted.However, identifying agents that accomplish the task of reactivatinglatent virus is difficult. Moreover, in vivo systems for reactivating alatent viral infection of difficult to manipulate or maintain acontrolled system such that endogenous cytokines do not affect theoutcome. Accordingly, an in vitro system for screening agents thatreactivate latent viruses is need. Disclosed is a method of screeningfor a composition that activates a cell latently infected by a virus;the method comprising the steps of: a) creating a latently infectedcell; b) exposing the cell to a test composition; and c) determining ifthe latently infected cell becomes active. It is understood and hereincontemplated that the latently infected cell can be made by the methodsdisclosed herein.

It is further understood that the in addition to screening for an agentthat activates a latently infected cell, disclosed herein are methods ofscreening for an agent that reactivates a latent virus in a latentlyinfected cell. Thus, disclosed are methods of screening for acomposition that reactivates latent virus in a cell latently infected bythe virus; the method comprising the steps of: a) creating a latentlyinfected cell; b) exposing the cell to a test composition; and c)determining if the virus in the latently infected cell becomes active.It is further understood that the disclosed methods of screening for anagent that reactivates a latent virus or activates a latently infectedcell are not mutually exclusive methods and the same screen can arriveat both results.

The determination of cellular activation can be achieved through anymeans known in the art for determining cellular proliferation. Forexample, cellular activity can be measured by flow cytometry through theuse of Carboxyfluorescein succinimidyl ester (CFSE), CD25, Ki-67, CD44.B220, CD69, loss of CD62L, or Propidium iodide. Additionally, cellularactivation can be measured by 3H-Thymidine incorporation. Viralreactivation can be determined by any measuring technique known in theart, including but not limited to flow cytometry through the use ofDsRed or HIV Gag p24, or using DNA measuring techniques such asquantitative PCR and methods that include the presence of a reportergene within the virus genome, such as luciferase, beta galactosidase andGFP can also be utilized.

Further disclosed is a composition identified by the screening methoddescribed above. In one example, the cell can be further exposed toCD3/CD28 antibodies during step b). The cell can also be exposed to PHAduring step b). The virus can be any virus capable of producing latentlyinfected cells. This includes, but is not limited to, retroviruses suchas HIV-1, HIV-2, SIV, XMRV, HTLV-1, HTLV-2, HTLV-3 and HTLV-4. The viruscan also be HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8, hepatitisC, or hepatitis B.

Also disclosed is an assay for determining a composition capable ofactivating a cell latently infected with a virus or reactivating alatent virus, the assay comprising an in vitro population of cellslatently infected with a retrovirus, wherein at least 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60%, or more of the cells are latently infected,and wherein the cell population is stable.

C. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include a cell assay of latentcells including cytokines or antibodies to aid activation orreactivation (e.g., IL-7, IL-2, αCD3/αCD28, IL-1, IL-10, IL-12, IL-15,IL-6, TNF-α, TGF-β, IFN-α and IFN-β), antibodies or other reagents tovisualize the assay results (e.g., CFSE, CD25, Ki-67, CD44. B220, CD69,loss of CD62L, Propidium iodide, and/or p24) and instructions forutilizing the components. Furthermore, the kits can include frozen andexpanded latently infected cells. Thus, for example, disclosed hereinare kits comprising a latently infected CD4 T cell, CFSE, and IL-7. Itis understood and herein contemplated that any antibodies supplied withthe kit can be modified to incorporate a detectable label.

As used herein, a label can include a fluorescent dye, a member of abinding pair, such as biotin/streptavidin, a metal (e.g., gold), or anepitope tag that can specifically interact with a molecule that can bedetected, such as by producing a colored substrate or fluorescence.Substances suitable for detectably labeling proteins include fluorescentdyes (also known herein as fluorochromes and fluorophores) and enzymesthat react with colorometric substrates (e.g., horseradish peroxidase).The use of fluorescent dyes is generally preferred in the practice ofthe invention as they can be detected at very low amounts. Furthermore,in the case where multiple antigens are reacted with a single array,each antigen can be labeled with a distinct fluorescent compound forsimultaneous detection. Labeled spots on the array are detected using afluorimeter, the presence of a signal indicating an antigen bound to aspecific antibody.

Fluorophores are compounds or molecules that luminesce. Typicallyfluorophores absorb electromagnetic energy at one wavelength and emitelectromagnetic energy at a second wavelength. Representativefluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS;4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein;5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT);5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-Imethylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; AcidFuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs—AutoFluorescentProtein—(Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™;Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™;Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X;Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate;APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R;Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA;ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue FluorescentProtein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst);bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy492/515;Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FLATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-Xconjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE;BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein;Calcein Blue; Calcium Crimson-; Calcium Green; Calcium Green-1 Ca²⁺ Dye;Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green-C18 Ca²⁺;Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); CascadeBlue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (CyanFluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A;Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; CoumarinPhalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan;Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP;cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; DansylCadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3′DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD-Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS;DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight;Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline);FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3;Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald;Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted(rsGFP); GFP wild type′ non-UV excitation (wtGFP); GFP wild type, UVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; LysoTracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso TrackerRed; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensorYellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red;Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange;Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; MaxilonBrilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker GreenFM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane;Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green PyronineStilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline;Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; OregonGreen™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; PacificBlue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-I PRO-3; Primuline;Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T;Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™(super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; StilbeneIsothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein;SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange;Spectrum Red; SPQ (6-methoxy-N-(3 sulfopropyl) quinolinium); Stilbene;Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOXGreen; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); TexasRed™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine RedR; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3;YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes);semiconductor nanoparticles such as quantum dots; or caged fluorophore(which can be activated with light or other electromagnetic energysource), or a combination thereof.

A modifier unit such as a radionuclide can be incorporated into orattached directly to any of the compounds described herein byhalogenation. Examples of radionuclides useful in this embodimentinclude, but are not limited to, tritium, iodine-125, iodine-131,iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13,fluorine-18. In another aspect, the radionuclide can be attached to alinking group or bound by a chelating group, which is then attached tothe compound directly or by means of a linker. Examples of radionuclidesuseful in the apset include, but are not limited to, Tc-99m, Re-186,Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62.Radiolabeling techniques such as these are routinely used in theradiopharmaceutical industry.

The radiolabeled compounds are useful as imaging agents to diagnoseneurological disease (e.g., a neurodegenerative disease) or a mentalcondition or to follow the progression or treatment of such a disease orcondition in a mammal (e.g., a human). The radiolabeled compoundsdescribed herein can be conveniently used in conjunction with imagingtechniques such as positron emission tomography (PET) or single photonemission computerized tomography (SPECT).

Labeling can be either direct or indirect. In direct labeling, thedetecting antibody (the antibody for the molecule of interest) ordetecting molecule (the molecule that can be bound by an antibody to themolecule of interest) include a label. Detection of the label indicatesthe presence of the detecting antibody or detecting molecule, which inturn indicates the presence of the molecule of interest or of anantibody to the molecule of interest, respectively. In indirectlabeling, an additional molecule or moiety is brought into contact with,or generated at the site of, the immunocomplex. For example, asignal-generating molecule or moiety such as an enzyme can be attachedto or associated with the detecting antibody or detecting molecule. Thesignal-generating molecule can then generate a detectable signal at thesite of the immunocomplex. For example, an enzyme, when supplied withsuitable substrate, can produce a visible or detectable product at thesite of the immunocomplex.

As another example of indirect labeling, an additional molecule (whichcan be referred to as a binding agent) that can bind to either themolecule of interest or to the antibody (primary antibody) to themolecule of interest, such as a second antibody to the primary antibody,can be contacted with the immunocomplex. The additional molecule canhave a label or signal-generating molecule or moiety. The additionalmolecule can be an antibody, which can thus be termed a secondaryantibody. Binding of a secondary antibody to the primary antibody canform a so-called sandwich with the first (or primary) antibody and themolecule of interest. The immune complexes can be contacted with thelabeled, secondary antibody under conditions effective and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes can then be generally washed to removeany non-specifically bound labeled secondary antibodies, and theremaining label in the secondary immune complexes can then be detected.The additional molecule can also be or include one of a pair ofmolecules or moieties that can bind to each other, such as thebiotin/avadin pair. In this mode, the detecting antibody or detectingmolecule should include the other member of the pair.

Other modes of indirect labeling include the detection of primary immunecomplexes by a two step approach. For example, a molecule (which can bereferred to as a first binding agent), such as an antibody, that hasbinding affinity for the molecule of interest or corresponding antibodycan be used to form secondary immune complexes, as described above.After washing, the secondary immune complexes can be contacted withanother molecule (which can be referred to as a second binding agent)that has binding affinity for the first binding agent, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (thus forming tertiary immune complexes).The second binding agent can be linked to a detectable label orsignal-generating molecule or moiety, allowing detection of the tertiaryimmune complexes thus formed. This system can provide for signalamplification.

D. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

The compositions found by the methods disclosed herein which can be usedfor treating HIV by, for example, causing latently infected cells toreactivate, can be administered in vivo in a pharmaceutically acceptablecarrier. By “pharmaceutically acceptable” is meant a material that isnot biologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the nucleic acid or vector,without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,including topical intranasal administration or administration byinhalant. As used herein, “topical intranasal administration” meansdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

(1) Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Formulations fortopical administration may include transdermal patches. Coated condoms,gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Compositions for parenteral, intrathecal or intraventricularadministration may include sterile aqueous solutions which may alsocontain buffers, diluents and other suitable additives.

In addition to such pharmaceutical carriers, cationic lipids may beincluded in the formulation to facilitate uptake. One such compositionshown to facilitate uptake is Lipofectin (BRL, Bethesda Md.).

E. Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Example 1

a) Materials and Methods

(1) Reagents

The following reagents were obtained through the AIDS Research andReference Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2from Dr. Maurice Gately, Hoffman-La Roche Inc. (Lahm et al. 1985);integrase Inhibitor (118-D-24) (Svarovskaia et al. 2004); and MonoclonalAntibody to HIV-1 p24 (AG3.0) from Dr. Jonathan Allan (Simm et al.1995).

(2) T Cells

Peripheral blood mononuclear cells were obtained from Leukopaks fromunidentified, healthy donors. Naïve CD4+ T cells were isolated by MACSmicrobead negative sorting using the naïve T cell isolation kit (MiltenyBiotec, Aurburn, Calif.). The purity of the sorted populations wasalways higher than 95% with a phenotype CD4⁺CD45RA⁺CD45RO⁻CCR7⁺CD62L⁺CD27⁺.

Naïve T cells were primed with beads coated with anti-CD3 and anti-CD28(Dynal/Invitrogen, Carlsbad, Calif.) as previously described (Messi etal. 2003). Proliferating cells were expanded in medium containing 30IU/ml IL-2, replacing medium and IL-2 each 2 days.

(3) Virus Generation and Viral Infection

DHIV viruses were produced by transient transfection of HEK293T cells bycalcium phosphate-mediated transfection (Zhu et al. 2001). To normalizeinfections, p24 was analyzed in virus-containing supernatants by ELISA(ZeptoMetrix Corporation, Buffalo, N.Y.). Cells were infected byspinoculation: 1×10⁶ cells were infected with 500 ng/ml of p24 during 2hours at 2900 rmp and 37° C. in 1 ml.

LTR mutants were generated by mutagenesis in DHIV using Quickchange IIXL (Startagen, Cedar Creek, Tex.). Mutations were confirmed bysequencing (See Supplementary Methods on line for the list of primersused).

(4) Flow Cytometry Analysis

To phenotype the cells, 2.5×10⁵ cells were stained with the followingmAbs: phycoerythrin-conjugated (PE)-anti-CD4, PE-anti-CCR5,PE-anti-CD45RO, PE-anti-CD27, fluorescein isothiocyanate-conjugated(FITC)-anti-CCR7, FITC-anti-CD45RA or PE-anti-CXCR4 (Caltag, Burlingame,Calif.) followed by flow cytometric analysis in a FACSCalibur using theCell Quest (Becton Dickinson, Mountain View, Calif.).

To assess intracellular p24-gag expression, 5×10⁵ cells were fixed andpermeabilized with Citofix/Cytoperm during 30 min at 4° C. (BDBiosciences, San Diego, Calif.). Cells were washed with Perm/Wash Buffer(BD Biosciences) and stained with 1:40 dilution of anti-p24 antibody(AG3.0) in 100 μl of Perm/Wash Buffer during 30 min at 4° C. Cells werewashed with Perm/Wash Buffer and incubated with 1:100 Alexa Fluor 488goat anti-mouse IgG (H+L) in 100 μl of Perm/Wash Buffer during 30 min at4° C. Cells were washed with Perm/Wash Buffer and samples were analyzedby flow cytometry. Forward versus side scatter plots were used to definethe live population. In all the experiments, HIV p24-gag stainingregions were set with uninfected cells treated in parallel.

Apoptosis was evaluated by simultaneous determination ofphosphatidylserine (PS) exposure and mitochondrial membrane potential(ΔΨ_(m)) in the same cells as previously described (Gomez-Benito et al.2005).

(5) Reactivation Assays

2.5×10⁵ cells were reactivated with beads coated with anti-CD3 andanti-CD28 during 72 hours in the presence of IL-2 (1 bead per cell).

For inhibition studies, cells were preincubated with the indicatedinhibitor for 2 hr before stimulation (See Supplementary Methods on linefor the concentration of each inhibitor or activator).

(6) Integration Analysis

Genomic DNA from 10⁶ was isolated with the DNeasy Tissue Kit (Quiagen,Valencia, Calif.). 250 ng of genomic DNA were subjected to quantitativeAlu-LTR PCR for integrated provirus as previously described (Vandegraaffet al. 2001, Butler et al. 2001, Dehart et al. 2005). (7) StatisticalMethods

Statistical analyses were performed with SPSS12.0 for Windows (SPSSInc., Chicago, Ill.). Two-tailed Paired-Samples T test analysis was usedto calculate the p value (α=0.05). Error bars in box-plots representrange.

b) Results

(1) A Novel Ex Vivo Paradigm to Study HIV-1 Latency

In order to recapitulate the generation of memory cell ex vivo, human,primary naïve CD4⁺ T cells were isolated using negative selection(Miltenyi Biotec, Auburn, Calif.; FIG. 1). The naïve cells were thenprimed toward differentiation into non-polarized (NP), T helper-1 (Th1)or T helper-2 (Th2) as previously described (Messi et al. 2003).

Phenotypic analysis confirmed the nature of the populations obtained invitro (FIG. 6). In vivo, memory CD4⁺ T cells fall into two maincategories, central memory (T_(CM)) and effector memory (T_(EM)). Thetranscriptional profile of in vivo T_(CM) closely resembles that of invitro T cells stimulated in NP conditions (Messi et al. 2003, Rivino etal. 2004). Specifically, both T_(CM) and NP cells are characterized bysimultaneous expression of CCR7 (a homing receptor for secondarylymphoid tissues) and CD27 (a coactivation molecule) (Messi et al. 2003,Rivino et al. 2004). Expression of CCR7 and CD27 is also found on naïvecells, but is absent in T_(EM). The analysis of CCR7 and CD27 expressionin the cells was conducted at 0 (naïve), 7, 14 and 21 days after initialactivation (FIG. 6). As expected, naïve CD4⁺ T cells expressed highlevels of CCR7 and CD27, as did cells primed in NP conditions. Incontrast to NP, priming under Th1- and Th2-polarizing conditions led toloss of CCR7 and CD27 expression, and generated populations withphenotypes that were characteristic of both T_(EM) cells and T_(CM)cells (FIG. 6).

At day 7, cells from NP, Th1 and Th2 conditions were exposed to virus. Aunique aspect of the model presented here is that the virus used in thismodel, DHIV (Andersen et al. 2006), has a small out-of-frame deletion inthe gp120-coding area that renders it defective in Env. To produce virusby transfection, HIV-1 Env is provided in trans in a separate plasmid(Challita-Eid et al. 1998). Due to the higher expression levels of CXCR4compared to CCR5 after the cells were activated (FIG. 6), a vector thatconsisted of the DHIV backbone pseudotyped with HIV-1_(LAI) (anX4-tropic virus) Env was produced. The engineered defect in Env in DHIVprecludes the production of infectious progeny after a single round ofinfection and thus the virus is unable to spread and cause massive celldeath, which obscures the emergence of latency in vitro.

Once infected, cells were kept in culture in the presence of IL-2, andinfection levels were estimated via intracellular expression ofp24^(Gag) at days 3 and 5 post infection (p.i.). Intracellular p24^(Gag)staining detects de novo produced viral Gag protein, indicative of aproductive viral infection. The maximal level of p24^(Gag) expressionwas observed 5 days p.i., and this level was highest in Th1 cells (FIG.2A, lower panels). Mock infected cultures displayed <0.5% background inintracellular p24^(Gag) staining.

At day 5 p.i., apoptosis levels were measured by flow cytometry.Positive staining for annexin V and low for DiOC₆(3) revealed thepresence of apoptotic cells (FIG. 2B). Apoptosis levels in DHIV-infectedNP and Th2 cells were similar to those in mock-infected cells. Incontrast, apoptosis in DHIV-infected Th1 cells was high. The higherlevel of apoptosis in Th1 (12.7% over mock) was in agreement with thehigher level of productive infection measured in these cells (14.5%)relative to other subsets.

In order to induce reactivation of potential latent viruses, at day 7p.i., cells were restimulated for 3 days in the presence of beads coatedwith αCD3 and αCD28 antibodies (FIG. 2C, CD3/CD28). As a negativecontrol, parallel cultures were incubated in the absence of beads (FIG.2C, untreated). Low levels of p24^(Gag+) cells were detected in theabsence of restimulation (FIG. 2C, upper panels). However, restimulationled to an increase in the percentage of p24^(Gag+) cells in all subsets(FIG. 2C, lower panels). Remarkably, levels of p24^(Gag+) cells afterreactivation were higher in NP cells than in Th1 or Th2.

The results shown in FIG. 2C correspond to a single blood donor (Donor1). To verify the generality of these findings in a broader population,further experiments were performed with 6 additional donors. Theresults, summarized in FIG. 2D, confirm that NP cells and, to a lesserdegree, Th2-polarized cells, can harbor high levels of HIV-1 latency,whereas Th1-polarized cells display lower levels of latency.

Quantitative Alu-PCR was used to evaluate the levels of viral infectionby a method that is independent of viral gene expression (Vandegraaff etal. 2001, Butler et al. 2001, Dehart et al. 2005). Quantitative Alu-PCRis specific for integrated viral DNA and detects latent and productiveinfections with the equal efficiency. Alu-PCR was performed at day 3p.i. for two Donors 1 and 2 (FIG. 2E). Alu-PCR results for Donor 1(square symbols) correspond to FIGS. 2A, C and D and show that thelevels of integrated viruses in all three cell subsets greatly exceededthe frequency of p24^(Gag) ⁺ cells that were observed at days 3 or 5p.i. These results indicate that this method leads to highly efficientgeneration of latently infected cells.

Th1 populations consistently contained lower levels of p24^(Gag+) cellsthan Th2 or NP upon reactivation. In addition, Th1 cells displayedlevels of infection by Alu-PCR that were roughly equivalent or higher(FIG. 2E) than those seen in Th2 or NP. Therefore, it appears that Th1cells are able to sustain higher levels of initial productive infection(i.e., p24^(Gag+); FIG. 2A, day 5 p.i), followed by higher levels ofapoptotic death, leading to less frequent latent infections.

Detection of viral gene expression can also be accomplished via reportermolecules, such as GFP, with high sensitivity and specificity (Jordan etal. 2003). To test whether the latency and reactivation system producesimilar results when using GFP as a reporter, parallel infections wereperformed with DHIV/X4 and DHIV-GFP/X4 (FIG. 7). In DHIV-GFP, nef hadbeen replaced by the enhanced green fluorescent protein gene (Jordan etal. 2003). Aside from the nef replacement with GFP, DHIV-GFP isidentical to DHIV. Most GFP-positive cells were also positive for p24,and vice versa, both during the initial infection and also afterreactivation (FIG. 7). A small population of cells that are positive forGFP but negative for p24 can also be appreciated. These cells are,presumably, early in the infection or reactivation process and have notbegun to produce viral late proteins. Therefore, intracellular p24detection and GFP fluorescence can be used interchangeably in order todetect viral gene expression in this latency and reactivation model.

(2) Signaling Pathways Involved in HIV-1 Reactivation in TCM

Previous studies on cells from infected patients showed that centralmemory CD4⁺ cells contain the highest frequency of HIV-1 DNA, on average10 times higher than that of effector memory cells (Brenchley et al.2004). Based on transcriptional profiles, cytokine production, surfacephenotype, and the ability to differentiate into effector memory cellsupon secondary antigenic challenge, NP cells are considered the in vitroequivalent of T_(CM) (Messi et al. 2003). Therefore, further studieswere focused on latent infection and reactivation of NP cells.

To begin to dissect potential signaling pathways leading to virusreactivation, a panel of known signaling inhibitors was tested.DHIV-infected NP cells were reactivated with αCD3/CD28, as shown in FIG.2C, in the presence or absence of pharmacological inhibitors (FIG. 3).As a control, and to confirm expectations that the observed latentinfections represent post-integration events, the integrase inhibitor,118-D-24, was tested (FIG. 3A). As expected, 118-D-24 did not have anynegative effect on viral reactivation.

One of the proximal events after activation of T cells through CD3 andCD28 is activation of the tyrosine kinase, Lck (see FIG. 8) (for areview, see (Kane et al. 2000)). Blocking Lck activation with PP2abrogated HIV-1 reactivation by about 96% (inhibition ofreactivation=(1-[p24% with αCD3/CD28 plus inhibitor−p24%untreated]/[p24% with αCD3/CD28−p24% untreated]×100; FIG. 3A). Lckactivation leads to PLCγ1 activation and production of the secondmessengers, diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3).DAG activates various isoforms of protein kinase C (PKC) (Isakov, 1987).Specifically in T cells, PKCθ leads to phosphorylation and degradationof IκB, with the subsequent release and nuclear translocation of thetranscription factor, NFκB (Bauer et al. 2000). The DAG-PKC-NFκBsignaling axis was probed by restimulating cells in the presence orabsence of the general PKC inhibitor, BIM; an inhibitor of the classicisoforms of PKC (α, β and γ), Gö6976 was also tested; as well asRottlerin, a specific inhibitor of PKCθ. None of these three compoundshad a negative effect on viral reactivation (FIG. 3A). The IκB kinasepeptide inhibitor (IKK inh) was used to confirm the previous result(Swaroop et al. 2001). When cells were reactivated in the presence ofIKK inh, the levels of viral reactivation were not affected.

DAG also activates the Ras guanyl-nulceotide-releasing protein (RasGRP)(Lin et al. 2001). RasGRP and many isoforms of PKC activate Ras, leadingto subsequent activation of the mitogen-activated protein (MAP) kinases,Erk1/2 (MEK), JNK, and p38. Inhibition of JNK or Erk1/2 with SP600125 orPD98059, respectively were used to probe the DAG-Ras-MAPK axis. NeitherSP600125 nor PD98059 inhibited viral reactivation. In contrast, aninhibitor of p38, SB202190, significantly diminished (66% inhibition;FIG. 3A) HIV-1 reactivation.

The other second messenger generated by PLCγ1, LP3, activatescalcineurin, which dephosphorylates and activates the transcriptionfactor, NFAT (Lin et al. 2001). To probe the IP3-dependent signalingcascade, restimulation was conducted in the presence or absence of thecalcineurin inhibitor, cyclosporine A (CsA). CsA completely abolished(99.9%) viral reactivation (FIG. 3A).

Stimulation through CD3/CD28 also involves recruitment and activation ofPI3K, leading to activation of the serine/threonine kinase, Akt (Frankeet al. 1995). PI3K is also involved in signal transduction downstream ofγc cytokine receptor engagement (FIG. 8). In order to ascertain thepossible contribution of PI3K, Wortmannin, a PI3k inhibitor was tested.Incubation with Wortmanin had no effect on HIV-1 reactivation.Leflunomide, the Janus-activated kinase-3 (JAK3) inhibitor, was alsotested to confirm the results with the γc cytokine-dependent pathway.Leflunomide also failed to block viral reactivation.

Signaling cascades in T cells can also be initiated through engagementof G protein-coupled receptors (GPCR; FIG. 8). GPCR signals typicallyconverge in the production of cyclic AMP (cAMP), which activates proteinkinase A (PKA), leading to the activation of the transcription factorCREB (for a review, see (Nordheim et al. 1994)). Cells were incubatedwith the PKA inhibitor, H-89, which did not have any effect on viralreactivation (FIG. 3A).

The above experiments with inhibitors were performed with two additionaldonors. The results with Donors 1, 3 and 4, summarized in FIG. 3B,denote striking similarities in the sensitivity of virus reactivation todrug inhibition across different individuals.

To complement the inhibitor studies, additional experimentation wasconducted using agonists for the above signaling pathways. Latentlyinfected cells were either left untreated or incubated with agonist. Asa positive control, cells were treated with αCD3/CD28. The ability ofthe agonist to promote viral reactivation was then evaluated bydetection of intracellular p24^(Gag) as shown above (FIG. 4).Phytohemagglutinin (PHA), a lectin that binds non-specifically tocarbohydrate moieties on surface glycoproteins and acts as a potentpolyclonal mitogen for T cells, was the first to be tested. PHAincubation efficiently reactivated viral gene expression (79%;reactivation efficiency=([p24% with agonist−p24% untreated]/[p24% withαCD3/CD28−p24% untreated]×100; FIG. 4A). the effect of PHA on T cellsare mediated by NFAT activation (FIG. 8). To confirm the role of NFAT inPHA-mediated reactivation, cells were co-incubated with PHA and the Lckinhibitor, PP2, or the calcineurin inhibitor, CsA. PHA stimulation inthe presence of PP2 or CsA resulted in extremely low viral reactivation(3% and 0%, respectively). These results are in complete agreement withthose from inhibitor studies and confirm the central role of NFAT inHIV-1 reactivation in memory T cells.

To activate the DAG-PKC-NFκB signaling axis, PMA and prostratin (bothdirect activators of PKC) were used separately. Neither compound wasable to reactivate viral gene expression (FIG. 4A).

Signaling downstream of IP3 involves an increase in intracellular levelsof calcium. To directly stimulate calcium influx, cells were incubatedwith Ionomycin, which had no effect on viral reactivation. However, acombination of PMA and Ionomycine was able to induce minor, butsignificant, levels of reactivation (4%).

In agreement with the lack of effect of H-89 (an inhibitor of PKA; FIG.3A), incubation of cells with the PKA activator, forskolin, failed toreactivate virus gene expression.

In tumor cell lines harboring integrated, latent HIV-1 TNF-α can induceviral gene expression through the activation of NFκB (Folks et al. 1989,Jordan et al. 2003, Osborn et al. 1989, Duh et al. 1989). The potentialrole of TNF-α in virus reactivation in latently infected memory T cellswas tested and it was found that TNF-α failed to induce any degree ofviral gene expression (FIG. 4A).

Inhibition of histone deacetylases (HDAC) with valproic acid (VA) haspreviously been shown to induce viral reactivation (Ylisastigui et al.2004, Simon et al. 1994, Lehrman et al. 2005). In the latent infectionsystem, however, incubation with VA failed to reactivate viral geneexpression (FIG. 4A).

As with the inhibitor studies, results with signaling pathway agonistswere very similar in three different donors (Donors 1, 3 and 4), asshown in FIG. 4B.

The studies above indicate that optimal HIV-1 reactivation in memory Tcells requires signaling events that involve, upstream, the tyrosinekinase, Lck, and, downstream, the transcription factor, NFAT.

(3) LTR Cis-Acting Elements Required for HIV-1 reactivation.

The HIV-1 latency model presented here uses molecularly cloned virus andrecapitulates a single virus replication cycle. Therefore, this systemallow us to ask which transcription factor binding sites in the viralpromoter are required for efficient reactivation. To that end, DHIVviral construct were engineered to contain mutations in specific regionsknown to regulate LTR-driven transcription (Tesmer et al. 1993, Sheridanet al. 1995, Ruocco et al. 1996, Perkins et al. 1994, Jones et al. 1986,d'Adda di Fafafna et al. 1995, Bohnlein et al. 1988). These mutationswere engineered in the U3 region of the 3′ LTR, such that the mutantpromoter was copied into the 5′ end of the virus after the first roundof reverse transcription. Mutants were constructed in the regions shownin FIG. 5A (See also the specific mutations in FIG. 9). NP cells wereinfected with mutant-promoter viruses, and cells were kept in vitro foran additional 7-day period. At this time point, immediately prior torestimulation, genomic DNA was isolated and quantitated viralintegration by Alu-PCR (blue numbers in FIG. 5B), to assess the levelsof latent infection prior to reativation. The cells were thenrestimulated with αCD3/CD28 and analyzed intracellular p24^(Gag)expression 3 days later.

As shown in FIG. 5B, mutation in the Sp1 sites abolished any ability oflatent viruses to be reactivated (0.3%; efficiency ofreactivation=([p24% of mutant with αCD3/CD28−p24% of mutant withoutαCD3/CD28]/[p24% of wt-virus with αCD3/CD28−p24% of wt-virus withoutαCD3/CD28]×100). Likewise, mutation of both κB/NFAT sites almostcompletely abolished reactivation (9.5%). Mutation of the AP-2 bindingsite led to a reactivation efficiency of 64%. Mutation of both NF-IL6 Iand NF-IL6 II binding sites, USF or TCF-1α had almost no effect (equalor higher than 85% reactivation efficiency).

The viral mutagenesis results together with the inhibitor and agoniststudies indicate that the transcription factors, NFAT and Sp1 areessential for reactivation in human NP memory CD4 T cells. Furtherdissection of signaling pathways and requirements for latency andreactivation can easily be pursued in the future using this ex vivosystem.

c) Discussion

HIV-1 latency reservoirs are small, but extremely long-lived. Latentinfection is associated with low-to-null levels of viral gene expressionand appears to be non-cytopathic. However, upon reactivation, latentviruses enter an active mode of replication in which they are fullycompetent for spread and induction of disease. The current thinking inthe field is that the use of a hypothetical drug that re-activateslatent viruses, in combination with present-day antiretroviral drugs, isthe desired approach toward viral eradication. However, the scarcity ofknown drugs that can safely be used for viral reactivation is alimitation. An additional limitation is the poor understanding of thedynamics between establishment of latency and reactivation, and thecellular and viral factors that govern these processes. This workdescribes the development of a novel method that recapitulates latentand productive viral infections in the laboratory. This method is easyto perform, powerful, and, most importantly, lends itself to molecularanalysis.

One key question about HIV-1 latency is what specific cell type(s) canharbor long-lived, latent proviruses. Previous work by several groups(reviewed in (Douek et al. 2003, Persaud et al. 2003)) indicates that invivo, quiescent memory T-cells constitute the most long-lived viralreservoir, whose decay constant ranges from months to years. Memorycells, in vivo, are subdivided into various subsets whose biology canfaithfully be recapitulated in vitro (Messi et al. 2003). The relativeabilities of NP, Th1 and Th2 cells to harbor latent viruses were testedand it was found that, while latent infections were induced in allsubsets, NP were consistently more permissive for latent infection andaccordingly less able to sustain productive infection. Conversely, Th1cells were exquisitely sensitive to productive infection, and latentinfection of these cells was significantly lower. It is tempting tospeculate that the higher permissiveness to productive infection of Th1cells was the cause of enhanced levels of apoptosis.

A unique aspect of the method presented here is that the virus isdefective in Env. When the virus is produced, Env is provided in trans.Thus, upon infection, viral particles contain the full proteincomplement of HIV-1, and are fully infectious and competent for entry,reverse transcription, integration, and viral gene expression. However,the engineered defect in Env precludes the production of infectiousprogeny. Cells undergoing productive infection die within 3-5 days dueto virus-mediated apoptosis and only uninfected and latently infectedcells survive after the first week in culture.

A second unique feature of this system is the intrinsic ability of thevirus to drive wild-type levels of gene expression. This is a crucialaspect of the model, as latent viruses in vivo, when reactivated, arefully capable of replicating and causing disease. This is an importantdistinction with previous models of latency in which lack of viral geneexpression was associated with mutations in the virus or in the hostcell (Duh et al. 1989, Kim et al. 2006, Chen et al. 1994, Cannon et al.1994, Butera et al. 1994) or with specific sites of virus integration inheterochromatin (Jordan et al. 2003).

HIV-1 latency appears to be related to intrinsic activation and/ordevelopmental characteristics of CD4+ T cells, rather than to thepresence of latency-promoting genes in the virus. Thus, it is importantto dissect, at a molecular level, the T-cell signaling pathway(s) thatunderlie the establishment, maintenance, and reactivation of latentinfections. In the present work, a three-prong approach is used todissect signaling events leading to reactivation in primary memorycells. Agonists and antagonists of cellular processes, and mutagenesisof viral cis-acting elements were used. The Sp1 and κB/NFAT promoterelements were critical toward reactivation.

Although Sp1 has been considered a ubiquitous and constitutivetranscription factor, an emerging body of evidence indicates that theactivity of Sp1 is regulated through the cell cycle (Vicart et al. 2006,Lacroix et al. 2002). Sp1 is phosphorylated and inactive in quiescentcells. Upon entry into the cell division cycle, PP2A dephosphorylatesSp1, which becomes active and tightly associated with the chromatin(Vicart et al. 2006, Lacroix et al. 2002). The finding that Sp1 isabsolutely required for reactivation of latent HIV-1 is in agreementwith the idea that a latently infected cell in vivo can be quiescent,and reactivation of the virus is concomitant with entry into the cellcycle.

The κB/NFAT binding sites, also a stringent requirement for viralreactivation, are not separable by mutagenesis because NFκB and NFATbind identical elements on the LTR (Giffin et al. 2003). The potentialroles played by NFAT and NFκB in reactivation are of paramountimportance in these studies, as it has recently been shown that navecells contain very low levels of NFATc1 and NFATc2, whereas memory cellscontain high levels of such transcription factors (Dienz et al. 2007).This explains why both nave and memory T cells rapidly induce IL-2(whose promoter contains a κB/NFAT binding site) transcription upon Tcell receptor ligation, but the responsible transcription factorsdiffer, being NFκB for nave cells, and NFAT for memory cells (Dienz etal. 2007). Therefore, in memory cells NFAT, but not NFκB, is essentialfor viral reactivation. The results from the inhibitor studies clearlyconfirm this prediction, as CsA incubation completely blockedreactivation whereas IKK Inh or PKC inhibitors had no effect. In furthersupport for the lack of a role of NFκB in viral reactivation in memorycells, agonists or stimuli that function through NFκB, such as PMA,prostratin and TNF-α, failed to induce any degree of reactivation.

Two agonists of NFAT activation, PHA and Ionomycin were tested. It isintriguing that PHA induced reactivation almost as efficiently asαCD3/CD28 treatment, while Ionomycin produced no detectablereactivation. PHA is a promiscuous mitogen that activates multiplepathways. However the observation that addition of CsA or PP2 completelyblocked reactivation by PHA further supports that the required signalingaxis dowstream of PHA is Lck-Calcineurin-NFAT.

Inhibition of p38 with SB202190 had a significant effect (66%inhibition) on viral reactivation. p38 participates in two signalingevents that can be relevant to viral reactivation (FIG. 8). Theclassical p38 activation pathway requires signaling throughDAG-RasGRP/PKC-Ras, whose inhibition did not affect reactivation. Inrecent years, an alternative p38 activation pathway has been described,which utilizes a scaffold protein known as Dlgh1 (Round et al. 2007).Dlgh1 is devoid of any known enzymatic activity, but can modify thesignaling emerging from TCR engagement, transmitted through ZAP70 andLck, to facilitate p38 activation and subsequent activation of NFAT in acalcineurin-dependent manner (see FIG. 8). Likely, this alternativepathway (Round et al. 2007) is the target of p38 inhibition on viralreactivation.

The results are similar, although with important differences, to thosereported earlier using a SCID-hu mouse model of HIV-1 latency (Brooks etal. 2003). The model by Brooks et al. and these results agree on therequirements for Lck and NFAT toward viral reactivation but disagree onthe requirement of NFκB (Brooks et al. 2003). A key difference in themodel proposed by Brooks et al. is the use of CD4 single-positivethymocytes, which can bare characteristics of naïve T cells rather thanmemory T cells.

In a Jurkat model of post-integration latency, it was found thatlatently infected cells frequently contained HIV-1 integrated in theproximity of alphoid repeat elements in heterochromatin (Jordan et al.2003). Reactivation of these latent viruses was accomplished with PMA orTNF-α. PMA and TNF-α failed to induce any detectable reactivation in thelatency system. The differences between these studies and the studies ofJordan et al. (Jordan et al. 2003). can be attributed to the use of aJurkat cell line. It is well known that Jurkat and primary T cellsshared some but not all T cell signaling pathways (Abraham et al. 2004).Since integration in this system is likely polyclonal, analysis of thecharacteristics of integration sites requires careful analysis.

Other means of inducing reactivation of latent proviruses have beenproposed, based on pharmacological modification of the “histone code”with histone deacetylase inhibitors, such as valproic acid (Lehrman etal. 2005). Valproic acid was incapable of inducing viral reactivation inthis model.

2. Example 2 Induction of HIV-1 Latency and Reactivation of PrimaryMemory CD4+T-cells a) Concentration of each Inhibitor or Activator

The concentration of each inhibitor or activator was as follows: 5 μMH-89, 50 μM PD98059, 250 nM Wortmannin, 15 nM bisindolylmaleimide II(BIM), 5 μM Rottlerin, 10 μM PP2, 50 μg/ml I kappa B Kinase InhibitorPeptide, Cell-Permeable (IKK inh.) and 50 μM Forskolin (Calbiochem, SanDiego, Calif.); 50 μM SB202190 and 125 ng/ml Leflunomide (AlexisBiochemicals, San Diego, Calif.); 10 nM Go6976 and 1 μM Prostratin (LCLaboratories, Woburn, Mass.); 25 ng/ml TNF-α (Prepotech Inc.); 10 ng/mlphorbol 12-myristate 13-acetate, 1 μM Ionomycin, 5 μg/ml L-PHA and 1 mMValproic Acid (Sigma, Saint Louis, Mo.); 500 ng/ml Cyclosporin A(Fluka/Sigma); 25 μM SP600125 (A.G. Scientific Int., San Diego, Calif.);and 20 μM Integrase Inhibitor (118-D-24).

b) Direct Mutagenesis of HIV-1 LTR

List of primers used to generate the different mutants. Nucleotidesmutated are indicated in bold

κB/NFAT-1 Forward 5′-tgacatcgagcttgctacaactcactttccgctggggac-3′ Reverse5′-gtccccagcggaaagtgagttgtagcaagctcgatgtc-3′ κB/NFAT-2 Forward5′-aagggactttccgctgctcactttccagggaggcg-3′ Reverse5′-cgcctccctggaaagtgageggaaagtccctt-3′ AP2 Forward5′-cttgctacaagggactttccatatgggactttccagggaggcgt-3′ Reverse5′-cgcctccctggaaagtcccatatggaaagtcccttgtagcaag-3′ Sp-1 Forward5′-ggggactttccagggattcgtggcctgttcgggactggttagtggcg ag-3′ Reverse5′-ctcgccactaaccagtcccgaacaggccacgaatccctggaaagtcc cc-3′ USF Forward5′-gtttgacagccgcctagcatttcatgaattcgcccgagagctgc-3′ Reverse5′-gcagctctcgggcgaattcatgaaatgetaggcggctgtcaaac-3′ TCF-1α Forward5′-gctgcatccggagtacgaattcaactgctgacatcgagc-3′ Reverse5′-gctcgatgtcagcagttgaattcgtactccggatgcagc-3′ NFIL6-I Forward5′-ttgacagccgcctagcatttaatcacgtggcc-3′ Reverse5′-ggccacgtgattaaatgctaggeggctgtcaa-3′ NFIL6-II Forward5′-cttcaagaactgctgacatcgagagctgtacaagggactttccgctg ggga-3′ Reverse5′-tccccagcggaaagtcccttgtacagctctcgatgtcagcagttctt gaag-

3. Example 3 a) The Effect of Homeostatic Proliferation on the LatentReservoir

Homeostatic proliferation is the ability of the immune system tomaintain normal T-cell counts, and to correct for deviations due toexpansion or depletion. Homeostatic proliferation is governed byextrinsic cellular signals, typically cytokines, in the absence ofantigenic stimulation. For CD4+ memory T cells, the γc-cytokine IL-7 iskey in governing this homeostasis. A role for IL-15, anotherγc-cytokine, has also been proposed. Homeostatic control involves boththe survival and the proliferation of memory CD4+ T cells. Therefore,quiescent memory cells harboring latent proviruses can enter the celldivision cycle. Reentry into the division cycle by quiescent memorycells can lead to concomitant viral reactivation. It is understood thatcell division can occur in the absence of viral reactivation. Thus,homeostatic proliferation can, actually, contribute to the expansion ofthe latent virus reservoir over time.

b) Latently Infected Memory Cells can Divide Without Reactivating Virus

Latently infected cells where stimulated with PMA+Ionomycin. Thistreatment induced vigorous cellular proliferation, as well as inductionof the activation markers, CD69 and CD25, but only induced a very smallamount of viral reactivation (4.2% of that obtained with αCD3/αCD28).This experiment can be repeated using physiological stimuli: cytokines.In addition, the experiments can be performed under conditions thatallow the skilled artisan to discern entry into the cell cycle and viralgene expression on a single-cell basis, using flow cytometry.

To perform these experiments, a latently infected population of cellswere generated and then the cells were reactivated in the presence ofIL-7 (plus IL-2, which is always maintained in culture to promotesurvival of cells). 48 or 72 hours later, cells were fixed,permeabilized and co-stained with two antibodies that recognizeintracellular p24 (for viral reactivation) and Ki-67. Ki-67 is a nuclearantigen whose expression is tightly regulated such that it is expressedin proliferating cells but absent in resting cells. As shown in FIG. 10,stimulation with IL-2+IL-7 induced 3.1+3.9=7.0% reactivation. Thedistribution of these cells into Ki-67-positive (dividing; 3.1%) andnegative (non-dividing; 3.9%) is similar (these populations arecircled). Therefore, remarkably, 56% of the cells that showed viralreactivation by IL-7 remained in a non-dividing status. This is thesecond stimulus found that can uncouple cell division and viralreactivation.

As a control for maximal reactivation αCD3/αCD28 was used. As expected,this treatment induced vigorous proliferation of most cells in culture(42.5%+39.5%=82%) and most or all the p24-positive cells fell within theKi-67-positive subset. Because αCD3/αCD28 (and its physiologicalcounterpart, TCR engagement) induced both potent activation andproliferation, this stimulus was not adequate to address how celldivision influences viral reactivation. Finally, because CD3/CD28reactivated 39.5+3.2=42.7% of the cells while IL2+1L7 only reactivated3.1+3.9=7.0%, there must be about 42.7-7.0=35.7% of cells stillharboring latent proviruses in the IL2+IL7 stimulated cultures.

c) The Ability of Latently Infected Memory Cells to Divide WithoutTriggering Viral Reactivation

These cells are present in the subset marked with a circle in FIG. 10.The cells from this subset can be sorted and then reactivate withαCD3/αCD28, to determine the presence of latent proviruses after cellsentered the cell cycle. Unfortunately, Ki-67 and p24 stainings requirecell fixation, which kills the cells. Therefore, to perform thisexperiment viable markers can be used. Instead of p24, a virus thatexpresses the DsRed fluorescent protein in place of nef is used. It isunderstood and contemplated herein that fluorescent markers arecompatible with the latency and reactivation system. After constructionof DHIV-DsRed, latently infected NP cells as are generated in the mannerdone with DHIV-GFP. Cells are cultured in IL-2, and one weekpost-infection, cells are changed to medium with IL-2+IL-7. Prior toIL-7 stimulation, cells are pulsed with CFSE, and at 72 hours poststimulation, cells are subjected to flow cytometric sorting, based ongreen and red fluorescence. Cells that have divided have their greenfluorescence (CFSE) diminished by at least half. Cells that containreactivated proviruses are red. The population that is low in CFSE andnegative for DsRed is purified. The high CFSE (non-dividing) and DsRednegative cells are also purified. These populations are assayed forintegrated viruses by Alu-PCR, and are cultured in IL-2 (negativecontrol) or with αCD3/αCD28 beads, to induce maximal reactivation. 48hours later, cells are analyzed by flow cytometry, for DsRed expression.The presence of DsRed positive cells in the αCD3/αCD28-treatmentindicates that latent proviruses are indeed present in the dividingpopulation, and indicate with what frequency. This indicates that, atleast in a certain subset of IL-7-treated cells, cell division isinduced without concomitant viral reactivation.

The converse scenario is that no DsRed positive cells are detected inthe dividing cells in the above experiment (again, this is theequivalent of the population in FIG. 10, except done with CFSE insteadof Ki-67).

d) Elucidating the Signaling Pathway that Mediates Viral Reactivation byIL-7

Interestingly, the signaling pathway initiated by IL-7+IL-2 isindependent of NFAT, because reactivation with IL-7+IL-2 is not blockedby cyclosporine A. The IL-7/IL-2 signaling pathway therefore representsan alternative means toward viral reactivation. Understanding theintracellular mediators of IL-7/IL-2 stimulation provides additionaltargets toward latent virus purging. Some of these mediators can beenzymes, such as kinases and phosphatases, which may be ameneable toagonistic or antagonistic drugs. It is also important to know theIL-7/IL-2 signaling because, as noted herein, this pathway is the basisfor homeostatic proliferation of memory cells, where an importantproportion of proliferating cells are not inducing viral reactivation.

The canonical IL-7 pathway is illustrated in FIG. 11. This pathway usesJAK3 and JAK1 proximally, and STATS distally. STATS binding sites arepresent within the LTR. A recent report suggests that three consensusSTATS binding sites can be found in the LTR. It was also shown thatectopic expression of STATS induced a 200-fold increase in LTR activityin cell lines, but only a 2-3 fold increase in primary cells. Toascertain the role of STATS in inducing LTR activity and viralreactivation, mutants in the three consensus STATS binding sites areconstructed. Individual site mutants are constructed as well as a doubleSite1-and-Site2 mutant and a triple mutant. Site 3 overlaps with theκB/NFAT binding site #2. Infection with these mutants is normal, as itwas for Sp1 binding site mutant. The efficiency of reactivation withIL2+1L-7 reveals the requirement of STATS sites for reactivation. As acontrol, an αCD3/αCD28 stimulation is used which is not expected torequire STATS. Further investigation of this signaling pathway includesEMSA, using synthetic oligonucleotides containing the predicted STATSsites, and ChIP. ChIP is performed as described herein and uses similarcontrols. As negative controls, STATS mutant viruses are used.

IL-7 stimulation induces activation of p38MAPK (a noncanonical IL-7dependent pathway). Induction of cellular proliferation by IL-7 wasreported to result in phosphorylation and activation of the p38α MAPK,and inhibition of p38α with SB203580 abrogated the proliferativeresponse. The results show that viral reactivation after TCR engagementis partially dependent on p38α activation (i.e., SB203580 induced about66% inhibition). Therefore, p38α is also a mediator for reactivation viaIL-7. Adding SB203580 simultaneously with IL-7 to latently infectedcells can block reactivation (FIG. 12).

However, lefluonomide, a Jak3 inhibitor, does not block reactivation inmemory cells and, therefore, contradicts the notion that IL-7 signalingthrough the canonical pathway enhances LTR activity via STATS. On theother hand, disclosed herein is evidence that p38α, when activateddownstream of αCD3/αCD28 stimulation, is required for viralreactivation. Thus, p38 is required for IL-7 reactivation. This is anexciting prospect because it means that p38α is a common signalingelement required for viral reactivation through two essentiallydifferent pathways, one being NFAT-dependent (αCD3/αCD28) and the otherone being independent (IL-7/IL-2). An explanation for this occurrence isthe subcellular localization of two different pools of p38. One pool iscytosolic (responding to IL-7 stimulation) and the other is localized tothe TCR signaling complex, in association with Dlgh1, Lck and ZAP-70.p38 activation in the latter pool is sensitive to Lck inhibition,whereas p38 in the former pool is not. Thus, the two putative pools ofp38 have different targets. The phoshporylation target for p38αemanating from TCR engagement is NFAT. However, the relevantphosphorylation target for p38α, downstream of IL-7 stimulation, is notNFAT, since viral reactivation by IL-7 is insensitive to CsA.

e) Differentiation of T_(CM) into T_(EM) in the Absence of AntigenicStimulation

Central Memory T cells (T_(CM)) cells can differentiate into effectormemory cells (T_(EM)) when IL-7 and IL-15 are combined with inflammatorycytokines, such as TNF-α, IL-6, IL-10 and IL-12. This differentiationprovides a mechanism for replenishing effector memory cells from a poolof central memory cells, in the absence of antigenic stimulation. The exvivo system disclosed herein can be used to investigate whether thistype of differentiation is sufficient to reactivate HIV-1 from latency.Differentiation of latently infected NP cells can be induced with eitherIL-7 or IL-15 plus a proinflammatory cytokine (TNF-α, IL-6, IL-10 orIL-12 can be utilized separately or in combination). Differentiation ofNP into T_(EM) cells can be analyzed by flow cytometry.

f) Effect of Bacterial Antigens and Inflammatory Mediators on the LatentReservoir

The immune system is subject to multiple antigenic threats, such asbacterial antigens. The presence of bacterial antigens can in many waysinduce inflammation directly or indirectly by inducing release ofinflammatory cytokines by immune cells. For example, HIV-1-induceddamage in the gut architecture leads to an increase in microbialtranslocation. This translocation leads to an increase on the levels ofbacterial lipopolysaccharide (LPS) and other bacterial products in theblood stream. This increase in bacterial products in the peripheryresults in reactivation of latent HIV-1. Disclosed herein, the effectsof different pathogen-associated molecular patterns (PAMPs), includingLPS, flagellin, lipoteichoic acid, peptidoglycan, and nucleic acidvariants normally associated with bacteria such as unmethylated CpGoligonucleotides are analyzed. These studies are extended to includereceptors for other PAMPS, such as those recognizing double-stranded RNA(dsRNA). Since these products can act directly on PAMP recognitionmolecules, and induce the release of pro-inflammatory cytokines andchemokines, select pro-inflammatory cytokines and chemokines areanalyzed for their ability to reactivate latent viruses.

PAMPs are mainly recognized by cells of the innate immune system, suchas monocyte/macrophages and dendritic cells, through the interactionwith a family of receptors called Toll-like receptors (TLRs 1-10).Specifically, TLR-4 recognizes LPS. Two different pathways are activatedafter TLR-4 ligation. The first one involves activation of NFκB and theproduction of inflammatory cytokines, such as IL-1, IL-6, TNF-α orTGF-β. The second one involves activation of IRF3, which leads to theproduction of IFN-β and interferon inducible genes. These cytokines canthen bind to receptors on latently infected T_(CM), to inducereactivation.

Monocytes are treated with TLR agonists (such as LPS, for TLR4), andtest the supernatant of treated monocytes on latently infected cells.When supernatants are able to induce viral reactivation, antibodiesagainst IL-1, IL-6, TNF-α, TGF-β and IFN-β are used to block individualcytokines and elucidate the involvement of particular cytokines.

Herein, the effects of PAMPs acting directly on latently infected cellsare disclosed. A growing body of evidence indicates that TLR expressioncan also be detected in T cells. However, no information is availableregarding the expression of these receptors on central memory CD4 Tcells. The expression of TLR receptors in NP cells is analyzed by flowcytometry. The expression of these receptors are confirmed on T_(CM)directly obtained from peripheral blood (instead of NP) using flowcytometry. Once identified the expression of TLRs in NP cells, similarstudies of reactivation are performed on latently infected NP cells asthe ones described above. TLR4 and its downstream signaling has beendealt with separately in more detail. To study the potential forreactivation, latently infected cells are incubated with specificagonists for each TLR, and reactivation are measured as % p24(+) cells.

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1. A method for creating a population of cells latently infected with avirus, the method comprising the steps of: a) isolating primary cells;b) priming the cells toward differentiation, wherein at least a portionof primary cells differentiate into non-polarized cells; c) exposing thenon-polarized cells of step b) to a virus defective in Env; therebycreating a population of cells latently infected with a virus andwherein the Env is provided in trans to the env defective virus whilethe virus is being grown, prior to exposure to the non-polarized cellsof step c.
 2. The method of claim 1, wherein the primary cell is a CD4+cell.
 3. The method of claim 2, wherein the primary CD4+ cell is a naïvecell.
 4. The method of claim 1, wherein the primary cell is amacrophage.
 5. The method of claim 1, wherein the virus is a retrovirus.6. The method of claim 5, wherein the retrovirus is selected from thegroup comprising HIV-1, HIV-2, SIV, XMRV, HTLV-1, HTLV-2, HTLV-3, orHTLV-4.
 7. The method of claim 1, wherein the virus is hepatitis B orhepatitis C.
 8. The method of claim 1, wherein the virus is a herpesvirus.
 9. The method of claim 8, wherein the herpes virus is selectedfrom the group consisting of HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7,and HHV-8.
 10. The method of claim 1, wherein at least 10% of thepopulation of cells is latently infected.
 11. The method of claim 1,wherein at least 20% of the population of cells is latently infected.12. The method of claim 1, wherein at least 30% of the population ofcells is latently infected.
 13. The method of claim 1, wherein at least40% of the population of cells is latently infected.
 14. A method ofscreening for a composition that activates a cell latently infected by avirus; the method comprising the steps of: a) creating a latentlyinfected cell using the method of claim 1; b) exposing the cell to atest composition; and c) determining if the latently infected cellbecomes active.
 15. A composition identified by the method of claim 14.16. The method of claim 14, wherein the cell is exposed to CD3/CD28antibodies during step b).
 17. The method of claim 14, wherein the cellis exposed to PHA during step b).
 18. The method of claim 14, whereinthe virus is a retrovirus.
 19. The method of claim 18, wherein theretrovirus is selected from the group comprising HIV-1, HIV-2, SIV,XMRV, HTLV-1, HTLV-2, HTLV-3, or HTLV-4.
 20. The method of claim 14,wherein the virus is hepatitis B or hepatitis C.
 21. The method of claim14, wherein the virus is a herpes virus.
 22. The method of claim 21,wherein the herpes virus is selected from the group consisting of HSV-1,HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, and HHV-8.
 23. A cell linecomprising non-polarized CD4+cells that have been latently infected witha virus.
 24. The cell line of claim 23, wherein the virus is aretrovirus.
 25. The cell line of claim 24, wherein the retrovirus isselected from the group comprising HIV-1, HIV-2, SIV, XMRV, HTLV-1,HTLV-2, HTLV-3, or HTLV-4.
 26. The cell line of claim 23, wherein thevirus is hepatitis B or hepatitis C.
 27. The method of claim 23, whereinthe virus is a herpes virus.
 28. The method of claim 27, wherein theherpes virus is selected from the group consisting of HSV-1, HSV-2, VZV,EBV, CMV, HHV-6, HHV-7, and HHV-8.
 29. The cell line of claim 23,wherein the virus is env-.
 30. A method of reactivating a cell latentlyinfected with virus, the method comprising activating NFAT in theabsence of NF-κB.
 31. The method of claim 30, wherein the virus is aretrovirus.
 32. The method of claim 31, wherein the retrovirus isselected from the group comprising HIV-1, HIV-2, SIV, XMRV, HTLV-1,HTLV-2, HTLV-3, or HTLV-4.
 33. The method of claim 30, wherein the virusis hepatitis B or hepatitis C.
 34. The method of claim 30, wherein thevirus is a herpes virus.
 35. The method of claim 34, wherein the herpesvirus is selected from the group consisting of HSV-1, HSV-2, VZV, EBV,CMV, HHV-6, HHV-7, and HHV-8.
 36. The method of claim 30, wherein thevirus is env-.
 37. The method of claim 30, wherein NFAT is activated inthe presence of CD3/CD28.
 38. The method of claim 30, wherein NFAT isactivated in the presence of CD3/CD28 and Sp1.
 39. A method ofreactivating a cell latently infected with virus, the method comprisingcontacting the cell with IL-7 in the absence of NFAT.
 40. The method ofclaim 39, wherein the virus is a retrovirus.
 41. The method of claim 40,wherein the retrovirus is selected from the group comprising HIV-1,HIV-2, SIV, XMRV, HTLV-1, HTLV-2, HTLV-3, or HTLV-4.
 42. The method ofclaim 39, wherein the virus is hepatitis B or hepatitis C.
 43. Themethod of claim 39, wherein the virus is a herpes virus.
 44. The methodof claim 43, wherein the herpes virus is selected from the groupconsisting of HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, and HHV-8. 45.The method of claim 39, further comprising contacting the cell with oneor more cytokines selected from the group consisting of IL-1, IL-2,IL-6, IL-10, IL-12, IL-15, TNF-α, TGF-β, IFN-α and IFN-β.
 46. The methodof claim 39, further comprising contacting the cell with apathogen-associated molecular patterns (PAMPs).
 47. The method of claim46, whereinthe PAMP is selected from the group consisting of LPS,flagellin, lipoteichoic acid, peptidoglycan, and unmethylated CpGoligonucleotides.
 48. A method of treating a subject with a latent viralinfection, the method comprising: a) exposing the subject to acomposition that reactivates cells latently infected with a virus; andb) treating the subject with an antiviral agent identified by the methodof claim
 14. 49. The method of claim 48, wherein the virus is HIV-1. 50.The method of claim 48, wherein the composition that reactivates celllatently infected with virus comprises one or more cytokines.
 51. Themethod of claim 48, wherein the antiviral agent is HAART.
 52. An assayfor determining a composition capable of reactivating a cell latentlyinfected with a retrovirus, the assay comprising an in vitro populationof cells latently infected with a retrovirus, wherein at least 20% ofthe cells are latently infected and wherein the cell population isstable.